KR101425048B1 - A composition for anti-virus comprising the extract or fraction of Daphne kiusiana as an effective ingredient - Google Patents

Abstract

The invention baekseohyang (Daphne The present invention relates to an antiviral composition comprising an extract or fraction thereof as an active ingredient, and more particularly, to a composition for an antiviral containing an extract or fraction thereof, and more particularly, to a composition for inhibiting the growth of natural killer (NK) cells by an immunotherapeutic cytokine such as interferon- -γ, and IFN-γ), and exhibits a potent antiviral activity, and thus has an excellent effect on viral diseases. Therefore, it can be effectively used for antiviral compositions.

Description

[0001] The present invention relates to an antiviral composition comprising an extract of Baucus orientalis or a fraction thereof as an active ingredient,

The invention baekseohyang (Daphne The present invention relates to an antiviral composition containing an extract or a fraction thereof as an active ingredient.

Many of the infectious diseases suffered by humans are caused by viruses, which cause many serious diseases such as rabies, smallpox, myelitis, hepatitis, yellow fever, immune deficiency, encephalitis and AIDS. Infectious diseases caused by such viruses are very contagious, such as colds, measles, mumps and chicken pox. They cause not only acute symptoms but also respiratory and digestive disorders. Measles and cytomegaloviruses cause congenital abnormalities, Viruses, known as tumor viruses, cause tumors and cancer in humans. Therefore, research on antiviral agents has been actively conducted for a long time. Antiviral drugs are oseltamivir (tamiflu), zanamivir (lilrenza), interferon, and immunoglobulin preparations, which weaken or exterminate the action of viruses that enter the human body. However, existing antiviral agents that have been used for the prevention and treatment of diseases caused by viral infections are problematic in their continued use due to the resistance and side effects of viruses caused by mutant viruses against these drugs. Therefore, there is a demand for the development of new antiviral agents derived from natural products which can solve the problems of the conventional antiviral agents. In particular, development of natural materials free from toxicity suitable for use as medicines and food additives is required.

Interferon-gamma (IFN-γ), on the other hand, is an immune system in which activated T cells and natural killer (NK) cells are generated in response to invasion of foreign substances such as viruses, parasites and tumor cells It is a natural protein. IFN-γ is secreted by T-helper 1 (Th1) cells, cytotoxic T, Tc cells, dendritic cells or NK cells in response to the presence of double helical RNA, Which is a type II (type II) interferon. IFN-γ is known to be a macrophage activator and has strong phagocytic activity. It has been studied to induce transcriptional changes of about 30 genes, and various immune and cellular responses (antigen presentation of macrophages , Increased lysosomal activity and activation in macrophages, inhibition of Th2 cell activation, increased expression of class I MHC molecules in normal cells, increased mobility of leukocyte cells, and increased NK cell activity). IFN-y aids the immune response by inhibiting viral replication in host cells, activating NK cells, increasing antigen presentation to lymph cells, and increasing host cell resistance to viral infection. When antigens are presented to matched T and B cells, they proliferate and remove foreign material in a strategic and specific way. In this regard, antigen presentation is a very important mechanism in the immune response.

NK cells are known to be involved in innate and acquired immune responses. NK cells mediate its effector function through cytotoxicity and cytokine production and function as cytotoxic effector cells mediated by ligands to receptors and target cells. NK-specific receptors for cytotoxicity include NKp46, referred to as natural cytotoxic receptors (NCRs) (Sivori, S. et al., J Exp Med , 186: 1129-1136, 1997), NKp30 (Pende, D. et al., J Exp Med. , 190: 1505-1516, 1999) and NKp44 (Cantoni, C. et al., J Exp Med , 189: 787-796, 1999). The ligands for NCRs include hemagglutinin (HA) of the influenza virus (IV), hemagglutinin-neuraminidase (HN) of the Sendai virus (SV) . Arnon, TI et al, Eur J Immunol, 31: 2680-2689, 2001) and a membrane-bound heparin sulfate proteoglycan (membrane-associated heparin sulfate proteoglycans) (Bloushtain, N. et al, J Immunol, 173: 2392. -2401, 2004). In addition, NK cells play an important role in helping the development of acquired immune responses through secretion of cytokines such as IFN-y (Stetson, DB et al., J Exp Med , 198: 1069-1076, 2003), it has been reported that IFN-y has antiviral activity in murine cytomegalovirus infection. NK cells are involved both in direct innate defense and in the formation of acquired immune responses. In some mouse models, NK cells inhibited tumor development and microbial infection. In particular, NK cells act as modulators in the early stages of mouse cytomegalovirus (MCMV) infection by directly killing viral infected cells and producing IFN-γ. The major pathway for IFN-y production in NK cells depends on the activation of protein kinase C (protein kinase C, PKC theta). Tassi and colleagues have reported that PKCθ is rapidly activated by the involvement of NK cell receptors that carry signals through immunoreceptor tyrosine-based activation motifs (ITAMs). Analysis of NK cells from PKCθ deficient mice revealed that PKCθ is an essential factor in ITAM-mediated IFN-γ secretion (Tassi, I. et al., Blood , 112: 4109-4116, 2008).

Phospholipase Cγ (phospholipase Cγ, PLCγ) is a very essential factor in IFN-γ secretion. The basal level of IFN-y was markedly decreased in PLCγ2-deficient NK cells, and unlike wild type cells, no stimulation by anti-NK1.1 resulted in increased IFN-γ secretion (Caraux, A. et al., Blood , 107: 994-1002, 2006). PLCγ2-deficient NK cells were severely impaired in the ability to produce IFN-γ or granulocyte-macrophage colony stimulating factor (GM-CSF), and PLCγ2 plays an important role in NK1.2-mediated cytokine production as well as in NKG2D (Regunathan, J. et al., J Immunol , 177: 5365-5376, 2006). Chain of the Fc receptor (FcRs) within the mouse that results in the tyrosine residue phosphorylation of the coupled adapter ITAMs (adapters < RTI ID = 0.0 > ITAMs), which again harbors spleen tyrosine kinase do. The kinase activates a number of lower signaling mediators including the linker for T cell activation, 76 kDa, PLC, PI3K and Erk kinase. Collectively, these signaling mediators have implicated gene transcription, a cell program for exocytosis of soluble granules that NK cells use to dissolve target cells, and produce proinflammatory chemokines and cytokines, particularly IFN-y (Tassi, I. et al., J Immunol , 175: 749-754, 2005). As described above, IFN-y has antiviral action, antiproliferative action and immunoregulatory action, and thus is used for treating hepatitis, various viral infectious diseases and cancer.

On the other hand, most viruses exhibit immune evasion strategies to protect themselves against host IFN responses. In particular, various mechanisms have been reported for suppression of IFN-y signal by virus. For example, Epstein-Barr viruses inhibit the expression of IFN-y receptor genes to block the antiviral IFN response (Morrison, TE, et al., Immunity 15: 787-799, 2001) It is known that IFN-y receptor 1 is inhibited by human tumor-induced herpesviruses (Li, Q., J Virol 81, 2117-2127, 2007). Therefore, there is a need to develop a substance capable of promoting secretion and activity of IFN, thereby increasing antiviral activity and immunological activity.

White Sheepskin ( Daphne kiusiana ) is an evergreen broad-leaved shrub of the dwarf- bellflower of the dwarf plant-plated neck, Daphne It is also called kiusiana and is known as white cherry tree. It is native to Korea and grows mainly at the foot of the beach, which is 50 ~ 1300m above sea level in Geoje Island, Jeolla Province, and Jeju Island. The height is about 1m. Leaves are alternate, oval or inverted. They are about 3 ~ 8 ㎝ in length and about 1.2 ~ 3.5 ㎝ in width. The edges are flat and glazed, and the bottom is narrow and continuous with short petiole. Flowers are dioecious and bloom in April to April, gathering at the ends of the branches and smelling the fragrance. The bract is broad lanceolate, peduncle is short, has white fine hairs, calyx has fine hairs, and is divided into 4 pieces. The fruit is a bowel shaped like an egg and it is about 8 ㎜ in length. It ripens in vermilion from May to June. The stem is upright and has a dark blue color and many branches form a covering type.

White sagittal flowers have been used in Oriental traditional medicine to treat endophthalmitis, toothache, rheumatism, pancreatitis and early breast cancer. Roots have been used to treat sore throat, leaves have been used to treat wounds, gout and chronic dermatitis. It is not clear yet (Lee, Seopheon, and Yeonglim, Seoul, pp.741-742, 1998). Anthocyanin-based and coumarin-based compounds were reported to be contained in white papers by a chemical composition study of white papers (Ishikura, N, I. Botanical Magazine , 88: 41-45, 1975; Nakabayashi, T, Yakugaku Zasshi , 74: 192-193, 1954). However, there has not yet been reported on the antiviral activity and the immune activity activity of the white sea side.

Accordingly, the inventors of the present invention have been studying to extract antiviral substances from natural extracts derived from plants, which can solve the problems of the antiviral agent of the prior art, and have found that the white leaf, stem, flower and root extract, The present inventors have completed the present invention by confirming that the secretion of interferon-gamma (IFN-y) is significantly increased in natural killer (NK) cells to have antiviral efficacy.

An object of the present invention is baekseohyang (Daphne The present invention also provides an antiviral composition comprising the extract or fraction thereof as an active ingredient.

In order to achieve the above object, the present invention baekseohyang (Daphne The present invention also provides a pharmaceutical composition for the prevention and treatment of viral diseases containing, as an active ingredient,

The present invention also provides a pharmaceutical composition for the prevention and treatment of viral diseases containing, as an active ingredient, an organic solvent fraction prepared by further extracting a white sea extract with an organic solvent.

The present invention also provides a health food for preventing and ameliorating a viral disease containing an extract of Baekseongbang or a fraction thereof as an active ingredient.

In addition, the present invention provides a feed additive for preventing and improving viral diseases containing an extract of Baekseongbang or a fraction thereof as an active ingredient.

Baekseohyang of the present invention (Daphne kiusiana extract or its fractions exhibit potent antiviral activity through secretion induction of interferon-γ (IFN-γ), an immune-related cytokine, in natural killer (NK) cells, It has an excellent effect and can be usefully used as an effective ingredient of a composition for antiviral use.

1 is a graph showing the amounts of secreted IFN-y after treating NK92 cells with methanol extracts of white leaves, stems, flowers and roots, n-hexane, ethyl acetate, butanol or water fractions thereof,
A: White backed methanol extract; And
B: Organic solvent fraction of white back methanol extract:
M: white methanolic methanol extract;
H: white sagittal n-hexane fraction;
E: white sheep ethyl acetate fraction;
B: white western butanol fraction;
W: white sea water fraction;
NC: negative control; And
PC: positive control.
FIG. 2 is a graph showing the amounts of secretion of IFN-y after NK92 cells were treated with fractions obtained by fractionating extracts of each of white-backed leaves, stems, flowers and roots:
A: Organic solvent fractions of methanol extract of white rye (DKCS: white seagrass; and DKCL: white seagrass);
B: n-hexane fraction of leaf of white backed cultivated sample; And
C: Ethyl acetate fraction of white backed reed sample leaves.
FIG. 3 is a graph showing the lowest activity concentration of IFN-gamma production ability of the white sea tangle extract.
FIG. 4 is a graph showing the expression of mRNA of IFN-? And? In NK92 cells treated with white-sided extract by RT-PCR.
FIG. 5 is a graph showing FACS analysis of the expression of cell surface material involved in the activity of NK92 cells treated with white sea tangle extract. FIG.

Hereinafter, terms used in the present invention will be described.

As used herein, the term "extract" has the meaning conventionally used in the art as a crude extract, but broadly includes the following fractions.

As used herein, the term "fraction" refers to an active fraction obtained by fractionating the activity of interest in the present invention using a solvent other than that used in the extraction.

The term "prophylactic ", as used herein, refers to any act that inhibits viral disease or delays progression upon administration of the composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions by which administration of the composition of the invention improves or alleviates symptoms of a viral disease.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

The term "individual" as used in the present invention means any animal such as a human, a monkey, a dog, a goat, a pig or a mouse having a disease in which symptoms of a viral disease can be improved by administering the composition of the present invention.

As used herein, the term "feed" refers to a material that maintains the life of a livestock and provides organic or inorganic nutrients necessary to produce milk, meat, eggs, fur, and the like.

Hereinafter, the present invention will be described in detail.

The invention baekseohyang (Daphne The present invention provides a pharmaceutical composition for the prevention and treatment of viral diseases containing an extract or a fraction thereof as an active ingredient.

The white backbone extract or its fractions can be obtained by various extraction methods known in the art.

The white sea tangle extract is preferably, but not necessarily, prepared by a manufacturing method comprising the following steps:

1) White Sphericity ( Daphne extracting by adding an extraction solvent to kiusiana);

2) cooling the extract of step 1) and filtering; And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above method, the white paper of step 1) may be used without limitation such as cultivated or marketed. The white sheet may be dried leaves, stalks, flowers or roots.

As the extraction solvent, any extraction solvent conventionally used in the art can be used, and it is preferable to use water, an alcohol or a mixture thereof. As the alcohol, C ₁ to C ₂ lower alcohols are preferably used, and as the lower alcohol, ethanol or methanol is preferably used. The extraction method is preferably, but not limited to, shaking extraction, reflux extraction, supercritical extraction or subcritical extraction. It is preferable that the extraction solvent is added by 2 to 20 times the amount of the dried white sachet. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 24 hours, but not always limited thereto. In addition, the number of times of extraction is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

Preferably, the fraction of the whitish extract is produced by a method comprising the step of adding an organic solvent to the whitish extract to prepare an organic solvent fraction.

In the above method, the organic solvent may be any extraction solvent conventionally used in the art, and may be an anhydrous or hydrated alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, n-propanol, iso- Acetone, chloroform, 1,3-butylene glycol, hexane, diethyl ether, or butyl acetate may be used as the organic solvent. The organic solvent may be hexane, ethyl acetate or butanol, but is not limited thereto.

It is preferable that fractions of each organic solvent layer are obtained by sequentially adding hexane, ethyl acetate, butanol, and water to the whitish extract in order from low to high polarity, and the fractions are preferably fractionated using a separating funnel, Not limited. Specifically, the white sagittal extract is suspended in distilled water, the hexane layer is separated by adding hexane, ethyl acetate is added to the remaining water layer to separate the ethyl acetate layer, and the butanol layer can be separated again by adding butanol to the remaining water layer. All fractions of each organic solvent layer can be used.

The white sea tangle extract or its fractions exhibit antiviral activity through induction of interferon-gamma (IFN-y) secretion in natural killer (NK) cells, but are not limited thereto.

The viral diseases include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, A virus selected from the group consisting of parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), human immunodeficiency virus (HIV) and hepatitis virus But is not limited thereto.

The present inventors investigated the antiviral activity of the white sea tangle extract or its fractions by measuring the induction activity of interferon-gamma (IFN-γ) secretion of white shark extract or fractions thereof by enzyme-linked immunosorbent assay assay, ELISA). As a result, it was shown that each white-backed leaf, stem, flower or root extract, or its fraction (n-hexane, ethyl acetate or butanol fraction) significantly increased the secretion of IFN-? In NK cells (Table 1 1 and FIG. 2), it was confirmed that the secretion of IFN-y was increased depending on the concentration of the white sea bream extract (see FIG. 3). The expression of IFN-y mRNA was also significantly increased in NK92 cells treated with white-western extract (see Fig. 4). On the other hand, FASC analysis of the effect of white sachet extract on the surface material expression of NK92 cells revealed that the expression of NKG2C and D that are involved in NK cell activity was significantly increased, and the expression of NKp44 (See Fig. 5).

IFN-y is a cytokine that is known to be a macrophage activator in natural killer (NK) cells and has strong phagocytic activity and is known to produce immune and cellular responses. Thus, it was confirmed that the extract of White Sag or extract thereof had a strong antiviral activity by inducing secretion of IFN-y.

Accordingly, it can be seen that the white sea tangle extract or the fraction thereof of the present invention can be effectively used as an effective ingredient of a pharmaceutical composition for the prevention or treatment of viral diseases.

The pharmaceutical composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions in addition to the above components.

Preferably, the pharmaceutical composition comprises 0.1 to 50 parts by weight of the whitish extract or fraction thereof, based on the total weight of the pharmaceutical composition, but is not limited thereto.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier other than the above-described effective ingredients. The pharmaceutically acceptable carriers to be included in the composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like . The composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may be varied depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Meanwhile, the oral dose of the composition of the present invention is preferably 0.0001 to 100 mg / kg (body weight) per day, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like. The administration route of the composition of the present invention is preferably determined depending on the type of disease to which it is applied.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The present invention also provides a method of preventing or treating a viral disease comprising administering to a subject a composition containing a pharmaceutically effective amount of a white sea bream extract or a fraction thereof as an active ingredient.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, method of administration, rate of elimination, severity of disease, and the like of a particular patient.

Preferably said animal is a vertebrate animal, more preferably an experimental animal such as a mouse, a rabbit, a guinea peak, a hamster, a dog or a cat, and most preferably an ape-like animal such as a chimpanzee or a gorilla.

The above-mentioned administration method may be oral or parenteral administration. In the case of parenteral administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, intramural injection, intracerebroventricular injection, Can be administered by injection.

The viral diseases include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, A virus selected from the group consisting of parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), human immunodeficiency virus (HIV) and hepatitis virus But is not limited thereto.

The white backbone extract or fraction thereof of the present invention exhibits a potent antiviral activity through secretion induction of IFN-y, which is an immuno-related cytokine in NK cells, and is thus effective for preventing or treating viral diseases Lt; / RTI >

The present invention also provides a health food for preventing and ameliorating a viral disease containing an extract of Baekseongbang or a fraction thereof as an active ingredient.

The viral diseases include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, A virus selected from the group consisting of parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), human immunodeficiency virus (HIV) and hepatitis virus But is not limited thereto.

The white sea extract of the present invention or a fraction thereof may be directly added, used in combination with other food or food ingredients, and suitably used according to a conventional method.

The health food of the present invention includes components that are ordinarily added at the time of food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings.

There is no particular limitation on the kind of the food. Examples of the food to which the white sea extract or its fractions can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Tea, a drink, an alcoholic beverage, and a vitamin complex, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the white sea tangle extract or its fractions of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, Glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the white sea extract or fraction thereof of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The white sea extract or fraction thereof of the present invention exhibits a potent antiviral activity through secretion induction of IFN-y, which is an immunological-related cytokine in NK cells, and has an excellent effect on viral diseases. Therefore, It can be used for food.

In addition, the present invention provides a feed additive for preventing and improving viral diseases containing an extract of Baekseongbang or a fraction thereof as an active ingredient.

The viral diseases include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, A virus selected from the group consisting of parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), human immunodeficiency virus (HIV) and hepatitis virus But is not limited thereto.

Since the feed additive has anti-viral efficacy, it can be prevented from viral diseases by continuously feeding it to poultry, domestic animals and the like, and viral diseases that have already occurred can be improved. Feeds can be classified into various categories according to their nutritional value, main ingredient, distribution, moisture content, processing type and the like, and the feeds can be used for forage, concentrated feed, supplementary feed, protein feed, starch feed, fat feed or fiber feed But is not limited thereto.

The feed additive of the present invention may contain 0.1 to 20% by weight of the whitening extract or its fraction, 0.001 to 0.01% by weight of lipase, 1 to 20% by weight of calcium phosphate, 0.1 to 10% by weight of the enzyme powder, 1 to 10% by weight of the enzyme powder, 0.1 to 10% by weight of the lactic acid bacterium, 0.01 to 10% by weight of the Bacillus culture, and 20 to 90% However, the present invention is not limited thereto, and if the white seaweed extract or its fractions are added in an effective amount, it can be used as the feed additive of the present invention.

The effective amount refers to an amount capable of preventing viral diseases or improving viral diseases that have already occurred by allowing them to continuously take poultry, livestock, and the like. Also, an amount that does not cause an adverse effect beyond the profit due to the addition is preferable.

The feed additive may additionally contain a carrier such as poultry and livestock. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like may be added. Various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added And the shape thereof may be a suitable state such as powder, granule, pellet, suspension and the like. When the feed additive of the present invention is supplied, it can be supplied to poultry, livestock and the like singly or mixed with feed.

The white backbone extract or fraction thereof of the present invention exhibits a potent antiviral activity through induction of secretion of IFN-y, which is an immuno-related cytokine in NK cells, and has an excellent effect on viral diseases. Therefore, It can be usefully used as an additive.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following examples and preparation examples illustrate the present invention specifically, and the content of the present invention is not limited by the examples and the production examples.

< Example  1> White Sack ( Daphne kiusiana ) Preparation of extract

<1-1> White tea leaf, stem, flower or root methanol extract

White Sheepskin ( Daphne kiusiana were harvested from Muan, Jeollanam - do, and 4 ℓ of methanol was added to 1 ㎏ of dried white leaves, stems, flowers or roots, and the filtrate supernatant was recovered by circulating at room temperature for 24 hours. After extraction, the solvent was concentrated under reduced pressure to obtain 118 g, 140 g, 80 g and 105 g of methanol extracts of white crustacean leaves, stems, flowers and roots, respectively.

< Comparative Example  1> Korean Won methanol extract

The Korean Won with reported anti-influenza virus activity ( Daphne genkwa ) (Korean Patent Application No .: 10-2009-0034132) was collected from Yongin, Gyeonggi-do and Korean Won-methanol extract was prepared according to the method of preparing the white methanolic extract of Example <1-1>.

< Example  2> White Sox Fraction  Produce

<2-1> White Sox n Hexane Fraction  Produce

Each of the white, leaf, stem, flower and root methanol extracts obtained in Example 1 was suspended in 1.5 L of distilled water, 1.5 L of n-hexane was added thereto, and the n-hexane soluble fraction and the water soluble fraction were separated . The n-hexane soluble fraction was filtered and concentrated under reduced pressure to obtain 31 g, 36 g, 21 g and 27 g of white shale leaves, stems, flowers and roots of n-hexane fractions respectively.

&Lt; 2-2 > White sheep ethyl acetate Fraction  Produce

The white leaves, stem, flower and root n-hexane fractions of Example <2-1> were separated, and 1.5 l of ethyl acetate was added to each of the remaining fractions. The fractions of white leaves, stem, flower and root ethyl acetate were 7 g, 8.4 g, 5 g and 6 g.

<2-3> White Sack Butanol Fraction  Produce

The white leaves, stem, flower, and root ethyl acetate fractions of Example <2-2> were separated, and 1.5 L of butanol was added to each of the remaining fraction portions, and 20 g of each of the white leaf, stem, flower and root butanol fractions were added, 24 g, 14 g and 18 g.

< Example  3> White sea tangle extract or its Fraction  Interferon-gamma interferon -γ, IFN -γ) secretion induction activity

<3-1> Natural killings natural killer , NK ) Cell culture

Interleukin-2 (IL-2) dependent natural killer (NK) cell line NK92 (human NK lymphoma) was purchased from the American Type Culture Collection (ATCC). NK92 cells were cultured with 20% fetal calf serum (FCS) (HyClone, Logan, UT), 2 mM L-glutamate, 100 / / ml penicillin, 100 / / ㎖ streptomycin (Life Technologies) and incubated with 100 U / ml IL-2 (Chiron, Emeryville, CA) (α-minimal essential medium (Life Technologies, Karlsruhe, Germany) at 37 ° C and 5% CO ₂ .

<3-2> Enzyme bound immunoabsorption analysis enzyme - linked immunosorbent assay , ELISA ) &Lt; / RTI &gt; Fraction  Interferon-gamma interferon -γ, IFN -γ) Secretion Inducibility

The cell culture of the NK92 cells cultured according to the above Example <3-1> was treated with 2 μg / ml white shark leaf, stem, flower or root extract and fraction (n-hexane, ethyl acetate, butanol or water fraction) After incubation at 37 DEG C for 18 hours, the cells were removed and supernatant was obtained. Quantification of human IFN-? Present in the cell culture of each NK92 cell was performed using commercially available monoclonal antibody (mAb) (Endogen) according to the manufacturer's protocol for enzyme-linked immunosorbent assay immunosorbent assay, ELISA). At this time, the Korean native extract and dimethylsulfoxide (DMSO) were used as positive (PC) and negative (NC) control groups, respectively. The quantification of IFN-y was expressed as the mean ± standard deviation of the values analyzed three times.

As a result, as shown in the following Table 1 and Fig. 1, about 2.4 to 2.6 ng / ml of IFN-y was secreted in each NK92 cell treated with white methanolic methanol extract (leaf, stem, flower or root). When the organic solvent fractions (n-hexane, ethyl acetate or butanol) except for the water layer, which is a fraction of white sagittal leaves, stems, flowers and root extracts, were treated, the fraction of normal-hexane contained about 1.7 to 1.8 ng / Acetate fraction of about 1.4 to 1.8 ng / ml and the butanol fraction of 1.6 to 1.9 ng / ml of IFN-y. In the case of the water fraction, about 0.2 to 0.4 ng / ml of IFN-y was secreted and showed almost no IFN-γ secretion induction. Cells treated with white sea tangle extract or fraction exhibited significantly higher IFN-y secretion than negative control (0.3 ng / ml) (Table 1 and Fig. 1). In addition, as shown in Fig. 2, IFN-y secretion effects of fractions of each of white sagittal leaf, stem flower, and root extract were confirmed (Fig. 2). Thus, it was confirmed that the white-backed leaf, stem, flower or root extract, or a fraction thereof, promotes secretion of IFN-y in NK cells.

Samples (2 [mu] g / ml) IFN-y secretion amount (ng / ml) White-backed leaves



Methanol extract 2.5 n-hexane fraction 1.7 Ethyl acetate fraction 1.4 Butanol fraction 1.6 Water fraction 0.4 White sagittal stem



Methanol extract 2.6 n-hexane fraction 1.8 Ethyl acetate fraction 1.7 Butanol fraction 1.8 Water fraction 0.5 White-backed flowers



Methanol extract 2.4 n-hexane fraction 1.8 Ethyl acetate fraction 1.7 Butanol fraction 1.7 Water fraction 0.3 Root of White Sack



Methanol extract 2.4 n-hexane fraction 1.7 Ethyl acetate fraction 1.8 Butanol fraction 1.9 Water fraction 0.3 KRW Methanol extract (positive control) 2.0 DMSO Negative control group 0.3

<3-3> ELISA  Interferon-gamma by concentration of white crust extract interferon -γ, IFN -γ) secretion induction activity

The amount of INF-γ secretion was determined according to the concentration of each of the methanol extracts of white russet according to the method of Example <3-2>. At this time, the methanol extracts were added at a concentration of 0.01, 0.1, 1 or 10 μg / ml, respectively.

As a result, as shown in Fig. 3, the white methanol methanol extract significantly increased the secretion amount of INF-? At a concentration of 1 占 퐂 / ml or more (Fig. 3). Thus, it was confirmed that the INF-γ secretion was increased depending on the concentration of the methanol extract of Baek-sang.

&Lt; 3-4 > The expression of genes involved in induction of interferon-gamma secretion Fraction  Check the effect

RT-PCR was performed to examine changes in mRNA expression associated with IFN-? And? Induction in NK92 cells. NK92 cells or lung epithelial cell line A549 were cultured for 12 hours with 2 ng of white sage extract. The cells were lysed at 15, 30, 45, 1, 4, and 12 hours after culturing and RNA was isolated according to a known protocol and analyzed for IFN-y mRNA expression by RT-PCR. The primers used were 5'-TCCCATGGGTTGTGTGTTTA-3 'for the forward primer and 5'-GTCAGGGTGCAGCCGG-3' for the reverse primer, and GAPDH was used as the internal standard. The GAPDH forward primer used was 5'-CCATCACCATCTTCCAGGAG- 3 'and the reverse primer was 5'-ACAGTCTTCTGGGTGGCAGT-3'.

The RT-PCR reaction was performed using the above primer pair and Taq polymerase (Takara, Shiga, Japan). Total RNA was isolated according to standard protocols and cDNA was synthesized using the AccuScript High Fidelity 1 ^st Strand cDNA Synthesis Kit (Stratagene) according to the manufacturer's instructions. 1 [mu] l of synthesized cDNA was used for 20 [mu] l PCR reaction consisting of 0.5 U ExTaq DNA polymerase, 1 x buffer, 1 mM dNTP mix (Takara) and the above primer pair. Amplification by PCR reaction was performed using GeneAmp PCR system 2700 (Applied Biosystems, Foster City, Calif., USA) under the following conditions; After 5 minutes at 94 占 폚, followed by 45 to 45 seconds at 94 占 폚, 45 seconds at 56 占 폚 and 1 to 25 minutes at 72 占 폚, the final extension reaction was carried out at 72 占 폚 for 7 minutes. PCR primers were designed using the Primer3 program and purchased from Bioneer (Daejeon, Korea). The PCR products were separated on 1.5% agarose gel, stained with ethidium bromide (EtBr) and visualized with a Gel Doc 2000 UV trans-illuminator (Bio-Rad Laboratories, Hercules, Calif., USA) (Bio-Rad Laboratories). Each sample was tested more than 3 times and representative data were presented.

As a result, as shown in Fig. 4, the expression pattern of IFN-y and IFN-a mRNA was examined in NK92 cells treated with white sagittal and A549 cells of lung epithelial cell line, and the expression of IFN- Was significantly increased after 3 hours in the treated NK92 cells, indicating that the response to IFN-γ induction in the white seaweed extract was induced within 3 hours at the transcription level. In A549 cells, whitish extract increased the expression level of IFN-α mRNA (FIG. 4).

< Example  4> Confirmation of effect on cell surface material of Baekseonghyang extract

In order to examine the changes of cell surface material involved in the activity of NK92 cells treated with white sagittal extract, fluorescence activated cell sorter (FACS) was used. At this time, NKG2A, C, and D acting as active ligands of NK cells, and NKp30, NKp44 and Nkp46 cell surface materials related to natural killer activity were analyzed.

As a result, as shown in Fig. 5, the NK92 cells treated with the white sea bream extract significantly increased the expression of NKG2C and D, which are involved in the activity of NK cells, and the expression of NKp44 associated with the natural killer activity of NK cells (Fig. 5).

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

Butanol fraction of white shark leaves 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of butanol fraction from white shark leaves

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of butanol fraction from white shark leaves

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of butanol fraction

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

Butanol fraction of white shark leaves 150 mg

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing of food

Foods containing the white sea bream extract of the present invention were prepared as follows.

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the white sage stem butanol fraction of the present invention was added to wheat flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of the white shrub butanol fraction of the present invention was added to the soup and the juice to prepare health promotion meat product, noodle soup and juice.

<2-3> Ground Beef  Produce

10 parts by weight of the white sage stem butanol fraction of the present invention was added to ground beef to prepare ground beef for health promotion.

<2-4> Dairy products ( dairy products )

5 to 10 parts by weight of the white sage stem butanol fraction of the present invention was added to milk and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The white shrub butanol fraction of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and white shale stem butanol fractions prepared above were blended in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(3 parts by weight), white sausage stem butanol fraction

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

(5%) of the white flowering butanol fraction of the present invention was uniformly blended with a sub ingredient such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% And packaged in small containers such as glass bottles and plastic bottles.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of the white rot flower butanol fraction of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 1 g of the peppery fraction of the present invention to 1,000 ml of apple or grape juice.

< Manufacturing example  4> Preparation of feed additive

The inventors of the present invention prepared a feed additive with the following composition using the whitish stem ethyl acetate fraction as an active ingredient.

<Composition of Feed Additive>

White sagittal stem ethyl acetate fraction: 0.1 to 20% by weight,

Lipase: 0.001 to 0.01% by weight,

Tribasic calcium phosphate: 1 to 20% by weight,

Vitamin E: 0.01 to 0.1% by weight,

Enzyme powder: 1 to 10% by weight,

Lactic acid bacteria: 0.1 to 10% by weight,

Bacillus culture medium: 0.01 to 10% by weight, and

Glucose: 20 to 90% by weight.

As described above, since the white sea bream extract or its fractions of the present invention have an excellent effect on antivirus, development of a medicament for the prevention and treatment of viral diseases or prevention and improvement of viral diseases and development of health food and feed additive Or treatment methods.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A composition for anti-virus comprising the extract or fraction          of Daphne kiusiana as an effective ingredient <130> 10p-11-05 <150> KR10-2009-0105439 <151> 2009-11-03 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Interferon-gamma forward primer <400> 1 tcccatgggt tgtgtgttta 20 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> interferon-gamma reverse primer <400> 2 gtcagggtgc agccgg 16 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 3 ccatcaccat cttccaggag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 4 acagtcttct gggtggcagt 20

Claims (11)

The extract of Daphne kiusiana as an active ingredient and the above white sea tangle extract is obtained by mixing at least one selected from the group consisting of white leaves, stem, flower or roots with water, a C1 to C2 lower alcohol or a mixed solvent thereof Wherein the pharmaceutical composition is for the prevention and treatment of viral diseases.
delete delete The pharmaceutical composition according to claim 1, wherein the lower alcohol is ethanol or methanol.
A pharmaceutical composition for the prevention and treatment of viral diseases containing hexane, ethyl acetate or butanol fraction prepared by extracting the white sea bream extract of claim 1 additionally with hexane, ethyl acetate or butanol as an active ingredient.
delete The method according to any one of claims 1 to 5, wherein the viral disease is selected from the group consisting of influenza virus, Influenza A virus subtype H1N1, avian influenza virus, Viruses such as rhinovirus, adenovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), and hepatitis virus Wherein the virus is infected by any one or more viruses selected from the group consisting of viruses.
WHAT IS CLAIMED IS: 1. A health food for preventing or ameliorating a viral disease containing as an active ingredient an extract of Baekseokgang or its hexane, ethyl acetate or butanol fraction.
9. The method of claim 8, wherein the viral disease is selected from the group consisting of influenza virus, Influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, A virus selected from the group consisting of coronavirus, parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), and hepatitis virus. Or a pharmaceutically acceptable salt thereof, for the prevention and the amelioration of a viral disease.
A feed additive for preventing and improving viral diseases containing an extract of Baekseongbang or its hexane, ethyl acetate or butanol fraction as an active ingredient.
11. The method of claim 10, wherein the viral disease is selected from the group consisting of influenza virus, Influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, A virus selected from the group consisting of coronavirus, parainfluenza virus, respiratory syncytial virus, herpesvirus (HSV), and hepatitis virus. Wherein the food additive is a feed additive for preventing and improving viral diseases.

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